Streptavidin

Streptavidin is a biotin binding protein from Streptomyces avidinii with an extraordinary of 10 M . This extreme affinity means that elution from immobilised biotin required 6 M guanidine hydrochloride at pH 1.5. By using 2-iminobiotin as a ligand rather than biotin the protein can be purified in a single chromatography step [48]. This illustrates that it may be worthwhile to consider an alternative ligand to what may at first appear to be the obvious natural choice. 

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In an extensive SFA study of protein receptor-ligand interactions, Leckband and co-workers [114] showed the importance of electrostatic, dispersion, steric, and hydrophobic forces in mediating the strong streptavidin-biotin interaction. Israelachvili and co-workers [66, 115] have measured the Hamaker constant for the dispersion interaction between phospholipid bilayers and find A = 7.5 1.5 X 10 erg in water. 

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... 

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... 

Fig. XV-4. Schematic drawing of four streptavidin molecules bound to biotinylated lipid in a monolayer above heavy water. The scattering length density for neutron reflectivity is shown at the side. (From Ref. 30.)...

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.)...

A monolayer of Streptavidin containing 1.75 mg of protein/m gives a film pressure of 0.070 erg/m at 15°C. Calculate the molecular weight of the protein, assuming ideal-gas behavior. 

Lee G U, Kidwell D A and Colton R J 1994 Sensing discrete streptavidin-biotin interactions with atomic force microscopy Langmuir 10 354... 

Figure Bl.20.10. Typical force curve for a streptavidin surface interacting with a biotin surface in an aqueous electrolyte of controlled pH. This result demonstrates the power of specific protein interactions. Reproduced with pennission from [81].

Direct measurement of the interaction potential between tethered ligand (biotin) and receptor (streptavidin) have been reported by Wong et al [16] and demonstrate the possibility of controlling range and dynamics of specific biologic interactions via a flexible PEG-tether. 

We assume in the following that the ligand is bound in a binding pocket of depth 6 —a = 7 A involving a potential barrier AU = 25 kcal/mol, similar to that of streptavidin (Chilcotti et al., 1995). We also assume that the diffusion coefficient of the ligand is similar to the diffusion coefficient of the heme group in myoglobin (Z) = 1 A /ns) as determined from Mofibauer spectra (Nadler and Schulten, 1984). 

Grubmiiller et al., 1996] Grubmiiller, H., Heymann, B., and Tavan, P. Ligand binding and molecular mechanics calculation of the streptavidin-biotin rupture force. Science. 271 (1996) 997-999... 

Both the AFM rupture experiments as well as our simulation studies focussed on the streptavidin-biotin complex as a model system for specific ligand binding. Streptavidin is a particularly well-studied protein and binds its ligand biotin with high affinity and specificity [51]. Whereas previous experiments (see references in Ref. [49]) and simulation studies [52] referred only to bound/unbound states and the associated kinetics, the recent AFM... 

In summary, our simulations provided detailed insight into the complex mcf hanisms of streptavidin-biotin rupture. They attribute the binding force... 

Fig. 6. Force profile obtained from a one nanosecond simulation of streptavidin-biotin rupture showing a series of subsequent force peaks most of these can be related to the rupture of individual microscopic interactions such as hydrogen bonds (bold dashed lines indicate their time of rupture) or water bridges (thin dashed lines).

S. Miyamoto and P. A. Kollman. Absolute and relative binding free energy calculations of the interaction of biotin and its analogs with streptavidin using molecular dynamics/free energy perturbation approaches. Proteins, 16 226-245, 1993. 

Miyamoto S and P A Kollman 1993a. Absolute and Relative Binding Tree Energy Calculations of the Interaction of Biotin and its Analogues with Streptavidin Using Molecular Dynamics/Free Energy Perturbation Approaches. Proteins Structure, Function and Genetics 16 226-245. 

An enzyme-amplified detection scheme, based on tire coupling of a streptavidin-alkaline phosphatase conjugate and biotinylated target sequences was then applied. The enzyme catalysed the hydrolysis of the elecn oiiractive a-naphthyl phosphate to a-naphtlrol this product is elecU oactive and has been detected by means of differential... 

Figure 3.7 The biotin-streptavidin system for enantioselective hydrogenation.

Fig. 4.5 Schematic representation of PD-ioop two homopyrimidine PNA openers binding to cioseiy positioned sites create one iarge DNA ioop to which an oiigonucieotide can bind. Such a compiex may be used to capture the DNA (e.g. via streptavidin beads) by empioying a biotinyiated oiigonucieotide.

Fig. 16 (a) Sequences of peptides pi6 and B2x-pi6. (b) Biotin-derivatised colibrils binding to a functionalised streptavidin with a functional group. Reproduced by permission of The Royal Society of Qiemistry from Kodama et al. [56]... 

Streptavidin with attached gold particles was added to mixtures of pl6 and B2x-pl6, starting with no biotin and increasing the molar ratio up to 1% of B2x-pl6. This increased the amount of streptavidin attached to fibrils, demonstrating that the amount of biotin present in the fibrils was dependent on this ratio. 

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