A streptavidin variant with slower biotin dissociation and increased mechanostability.
Ontology highlight
ABSTRACT:
SUBMITTER: Chivers CE
PROVIDER: S-EPMC2862113 | biostudies-literature | 2010 May
REPOSITORIES: biostudies-literature
Publications
A streptavidin variant with slower biotin dissociation and increased mechanostability.
Chivers Claire E CE Crozat Estelle E Chu Calvin C Moy Vincent T VT Sherratt David J DJ Howarth Mark M
Nature methods 20100411 5
Streptavidin binds biotin conjugates with exceptional stability but dissociation does occur, limiting its use in imaging, DNA amplification and nanotechnology. We identified a mutant streptavidin, traptavidin, with more than tenfold slower biotin dissociation, increased mechanical strength and improved thermostability; this resilience should enable diverse applications. FtsK, a motor protein important in chromosome segregation, rapidly displaced streptavidin from biotinylated DNA, whereas trapta ...[more]
Similar Datasets
Project description:It is currently unclear whether small molecules dissociate from a protein binding site along a defined pathway or through a collection of dissociation pathways. We report herein a joint crystallographic, computational, and biophysical study that suggests the Asp-128 --> Ala (D128A) streptavidin mutant closely mimics an intermediate on a well-defined dissociation pathway. Asp-128 is hydrogen bonded to a ureido nitrogen of biotin and also networks with the important aromatic binding contacts Trp-92 and Trp-108. The Asn-23 hydrogen bond to the ureido oxygen of biotin is lengthened to 3.8 A in the D128A structure, and a water molecule has moved into the pocket to replace the missing carboxylate interaction. These alterations are accompanied by the coupled movement of biotin, the flexible binding loop containing Ser-45, and the loop containing the Ser-27 hydrogen bonding contact. This structure closely parallels a key intermediate observed in a potential of mean force-simulated dissociation pathway of native streptavidin, where the Asn-23 hydrogen bond breaks first, accompanied by the replacement of the Asp-128 hydrogen bond by an entering water molecule. Furthermore, both biotin and the flexible loop move in a concerted conformational change that closely approximates the D128A structural changes. The activation and thermodynamic parameters for the D128A mutant were measured and are consistent with an intermediate that has traversed the early portion of the dissociation reaction coordinate through endothermic bond breaking and concomitant gain in configurational entropy. These composite results suggest that the D128A mutant provides a structural "snapshot" of an early intermediate on a relatively well-defined dissociation pathway for biotin.
| S-EPMC17525 | biostudies-literature
Project description:In vitro analysis of primary isolated adult cardiomyocyte physiological processes often involves optical imaging of dye-loaded cells on a glass substrate. However, when exposed to rapid solution changes, primary cardiomyocytes often move to compromise quantitative measures. Improved immobilization of cells to glass would permit higher throughput assays. Here, we engineer the peripheral membrane of cardiomyocytes with biotin to anchor cardiomyocytes to borosilicate glass coverslips functionalized with streptavidin. We use a rat cardiac myoblast cell line to determine general relationships between processing conditions, ligand density on the cell and the glass substrate, cellular function, and cell retention under shear flow. Use of the streptavidin-biotin system allows for more than 80% retention of cardiac myoblasts under conventional rinsing procedures, while unmodified cells are largely rinsed away. The adhesion system enables the in-field retention of cardiac cells during rapid fluid changes using traditional pipetting or a modern microfluidic system at a flow rate of 160 mL/min. Under fluid flow, the surface-engineered primary adult cardiomyocytes are retained in the field of view of the microscope, while unmodified cells are rinsed away. Importantly, the engineered cardiomyocytes are functional following adhesion to the glass substrate, where contractions are readily observed. When applying this adhesion system to cardiomyocyte functional analysis, we measure calcium release transients by caffeine induction at an 80% success rate compared to 20% without surface engineering.
| S-EPMC8280672 | biostudies-literature
Project description:Macromolecules tend to respond to applied forces in many different ways. Chemistry at high shear forces can be intriguing, with relatively soft bonds becoming very stiff in specific force-loading geometries. Largely used in bionanotechnology, an important case is the streptavidin (SA)/biotin interaction. Although SA's four subunits have the same affinity, we find that the forces required to break the SA/biotin bond depend strongly on the attachment geometry. With AFM-based single-molecule force spectroscopy (SMFS), we measured unbinding forces of biotin from different SA subunits to range from 100 to more than 400 pN. Using a wide-sampling approach, we carried out hundreds of all-atom steered molecular dynamics (SMD) simulations for the entire system, including molecular linkers. Our strategy revealed the molecular mechanism that causes a fourfold difference in mechanical stability: Certain force-loading geometries induce conformational changes in SA's binding pocket lowering the energy barrier, which biotin has to overcome to escape the pocket.
| S-EPMC7096159 | biostudies-literature
Project description:Novel site-specific attachment strategies combined with improvements of computational resources enable new insights into the mechanics of the monovalent biotin/streptavidin complex under load and forced us to rethink the diversity of rupture forces reported in the literature. We discovered that the mechanical stability of this complex depends strongly on the geometry in which force is applied. By atomic force microscopy-based single molecule force spectroscopy we found unbinding of biotin to occur beyond 400 pN at force loading rates of 10 nN/s when monovalent streptavidin was tethered at its C-terminus. This value is about twice as high than that for N-terminal attachment. Steered molecular dynamics simulations provided a detailed picture of the mechanics of the unbinding process in the corresponding force loading geometries. Using machine learning techniques, we connected findings from hundreds of simulations to the experimental results, identifying different force propagation pathways. Interestingly, we observed that depending on force loading geometry, partial unfolding of N-terminal region of monovalent streptavidin occurs before biotin is released from the binding pocket.
| S-EPMC6486461 | biostudies-literature
Project description:Atomic resolution crystallographic studies of streptavidin and its biotin complex have been carried out at 1.03 and 0.95?Å, respectively. The wild-type protein crystallized with a tetramer in the asymmetric unit, while the crystals of the biotin complex contained two subunits in the asymmetric unit. Comparison of the six subunits shows the various ways in which the protein accommodates ligand binding and different crystal-packing environments. Conformational variation is found in each of the polypeptide loops connecting the eight strands in the ?-sandwich subunit, but the largest differences are found in the flexible binding loop (residues 45-52). In three of the unliganded subunits the loop is in an `open' conformation, while in the two subunits binding biotin, as well as in one of the unliganded subunits, this loop `closes' over the biotin-binding site. The `closed' loop contributes to the protein's high affinity for biotin. Analysis of the anisotropic displacement parameters included in the crystallographic models is consistent with the variation found in the loop structures and the view that the dynamic nature of the protein structure contributes to the ability of the protein to bind biotin so tightly.
| S-EPMC3169315 | biostudies-literature
Project description:Accelerated molecular dynamics (aMD) simulation is employed to study the functional dynamics of the flexible loop(3-4) in the strong-binding streptavidin-biotin complex system. Conventional molecular (cMD) simulation is also performed for comparison. The present study reveals the following important properties of the loop dynamics: (1) The transition of loop(3-4) from open to closed state is observed in 200 ns aMD simulation. (2) In the absence of biotin binding, the open-state streptavidin is more stable, which is consistent with experimental evidences. The free energy (?G) difference is about 5 kcal/mol between two states. But with biotin binding, the closed state is more stable due to electrostatic and hydrophobic interactions between the loop(3-4) and biotin. (3) The closure of loop(3-4) is concerted to the stable binding of biotin to streptavidin. When the loop(3-4) is in its open-state, biotin moves out of the binding pocket, indicating that the interactions between the loop(3-4) and biotin are essential in trapping biotin in the binding pocket. (4) In the tetrameric streptavidin system, the conformational change of the loop(3-4) in each monomer is independent of each other. That is, there is no cooperative binding for biotin bound to the four subunits of the tetramer.
| S-EPMC4298722 | biostudies-literature
Project description:The strong non-covalent interaction between biotin and streptavidin places streptavidin-based assays, used by many laboratories, at an increased risk of interference by biotin. At present, a few manufacturers have developed fully automated anti-biotin interference methods, although compared with many detection platforms, these remain insufficient. Additionally, there is a need for more methods that can achieve fully automated anti-biotin interference. We sought to develop and evaluate a new biotin interference-resisting method based on a biotin-streptavidin chemiluminescence immunoassay. Streptavidin-coated magnetic microparticles (M) of different concentrations were prepared and tested for their biotin-resistance capabilities in an automated setting (Cobas e 601). The precision, accuracy, and detection capability were also assessed. Higher concentrations of M were found to have a stronger ability to resist biotin interference. A 2.16 mg/mL concentration of M was able to resist 500 ng/mL of biotin in samples while simultaneously having a relatively weak shielding effect on the optical signals. Moreover, the total precision and accuracy of this method, designated as M3, met acceptable standards. M3 has an improved ability to resist biotin interference, can achieve full automation, and its detection performance can meet the general laboratory quality requirements.
| S-EPMC9324022 | biostudies-literature
Project description:We recently reported on a potent synthetic agent, 135H11, that selectively targets the receptor tyrosine kinase, EphA2. While 135H11 possesses a relatively high binding affinity for the ligand-binding domain of EphA2 (Kd~130 nM), receptor activation in the cell required the synthesis of dimeric versions of such agent (namely 135H12). This was expected given that the natural ephrin ligands also need to be dimerized or clustered to elicit agonistic activity in cell. In the present report we investigated whether the agonistic activity of 135H11 could be enhanced by biotin conjugation followed by complex formation with streptavidin. Therefore, we measured the agonistic EphA2 activity of 135H11-biotin (147B5) at various agent/streptavidin ratios, side by side with 135H12, and a scrambled version of 147B5 in pancreatic- and breast-cancer cell lines. The (147B5)n-streptavidin complexes (when n = 2, 3, 4, but not when n = 1) induced a strong receptor degradation effect in both cell lines compared to 135H12 or the (scrambled-147B5)4-streptavidin complex as a control, indicating that multimerization of the targeting agent resulted in an increased ability to cause receptor clustering and internalization. Subsequently, we prepared an Alexa-Fluor-streptavidin conjugate to demonstrate that (147B5)4-AF-streptavidin, but not the scrambled equivalent complex, concentrates in pancreatic and breast cancers in orthotopic nude-mouse models. Hence, we conclude that these novel targeting agents, with proper derivatization with imaging reagents or chemotherapy, can be used as diagnostics, and/or to deliver chemotherapy selectively to EphA2-expressing tumors.
| S-EPMC8235110 | biostudies-literature
Project description:The thermodynamic and structural cooperativity between the Ser45- and D128-biotin hydrogen bonds was measured by calorimetric and X-ray crystallographic studies of the S45A/D128A double mutant of streptavidin. The double mutant exhibits a binding affinity approximately 2x10(7) times lower than that of wild-type streptavidin at 25 degrees C. The corresponding reduction in binding free energy (DeltaDeltaG) of 10.1 kcal/mol was nearly completely due to binding enthalpy losses at this temperature. The loss of binding affinity is 11-fold greater than that predicted by a linear combination of the single-mutant energetic perturbations (8.7 kcal/mol), indicating that these two mutations interact cooperatively. Crystallographic characterization of the double mutant and comparison with the two single mutant structures suggest that structural rearrangements at the S45 position, when the D128 carboxylate is removed, mask the true energetic contribution of the D128-biotin interaction. Taken together, the thermodynamic and structural analyses support the conclusion that the wild-type hydrogen bond between D128-OD and biotin-N2 is thermodynamically stronger than that between S45-OG and biotin-N1.
| S-EPMC2249767 | biostudies-literature
Project description:Target-activated chemical probes are important tools in basic biological research and medical diagnosis for monitoring enzyme activities and reactive small molecules. Based on the fluorescence turn-on mechanism, they can be divided into two classes: dye-based fluorescent probes and caged-luciferin. In this paper, we introduce a new type of chemical probe in which the fluorescence turn-on is based on controlled streptavidin-biotin binding. Compared to conventional probes, the streptavidin-biotin controlled binding probe has several advantages, such as minimal background at its "OFF" state, multiple signal amplification steps, and unlimited selection of the optimal dyes for detection. To expand the scope, a new synthetic method was developed, through which a wider range of analyte recognition groups can be easily introduced to construct the binding probe. This probe design was successfully applied to image and study secreted peroxynitrite (ONOO-) at the cell surface of macrophages where information on ONOO- is difficult to obtain. As the signals are generated upon the binding of streptavidin to the biotin probe, this highly versatile design can not only be used in fluorescence detection but can also be applied in various other detection modes, such as electrochemical and enzyme-amplified luminescence detection.
| S-EPMC5872805 | biostudies-other