A monovalent streptavidin with a single femtomolar biotin binding site

Howarth, Mark; Chinnapen, Daniel J.‐F.; Gerrow, Kimberly; Dorrestein, Pieter C.; Grandy, Melanie R.; Kelleher, Neil L.; El‐Husseini, Alaa; Ting, Alice Y.

doi:10.1038/nmeth861

Cited by 351 publications

(396 citation statements)

References 30 publications

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“…bR was purified from Halobacterium salinarum as previously described (22) and all mSA proteins and mutants were prepared as previously described (15,23), except that 2 mM DTT was added to all buffers when purifying Cys-containing bR and mSA mutants.…”

Section: Methodsmentioning

confidence: 99%

Measuring membrane protein stability under native conditions

Chang

Bowie

2013

Proc. Natl. Acad. Sci. U.S.A.

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The thermodynamic stability of proteins is typically measured at high denaturant concentrations and then extrapolated back to zero denaturant conditions to obtain unfolding free energies under native conditions. For membrane proteins, the extrapolations are fraught with considerable uncertainty as the denaturants may have complex effects on the membrane or micellar structure. We therefore sought to measure stability under native conditions, using a method that does not perturb the properties of the membrane or membrane mimetics. We use a technique called steric trapping to measure the thermodynamic stability of bacteriorhodopsin in bicelles and micelles. We find that bacteriorhodopsin has a high thermodynamic stability, with an unfolding free energy of ∼11 kcal/mol in dimyristoyl phosphatidylcholine bicelles. Nevertheless, the stability is much lower than predicted by extrapolation of measurements made at high denaturant concentrations. We investigated the discrepancy and found that unfolding free energy is not linear with denaturant concentration. Apparently, long extrapolations of helical membrane protein unfolding free energies must be treated with caution. Steric trapping, however, provides a method for making these measurements.membrane protein folding | steric trap M ethods to measure the thermodynamic stability of membrane proteins have largely followed methods developed for soluble protein folding (1). The fraction unfolded is first measured as a function of denaturant concentration (urea, guanidine-HCl, etc.), which in turn provides the unfolding free energy (ΔG U ) as a function of denaturant. The fraction unfolded, however, can be accurately measured only at high denaturant concentration, where the amount of unfolded protein is large enough-the so-called transition zone. Thus, obtaining a measure of the unfolding free energy in the absence of denaturant requires extrapolation from the transition zone. For chemical denaturation, the unfolding free energy is typically linearly dependent on the denaturant concentration in the transition zone, allowing a linear extrapolation back to zero denaturant. However, although there is now considerable experimental and theoretical validation of this approach for soluble proteins (2-4), the validity of these extrapolations is not clear for measuring stability of membrane proteins.Since the observation of Braiman et al. that bacteriorhodopsin (bR) can be refolded from an SDS-denatured state (5), SDS has been commonly used to study the folding of helical membrane proteins. The Booth laboratory has pioneered and extensively studied the refolding kinetics of bR from an SDS-denatured state (6-8). We introduced SDS unfolding to measure the thermodynamic stability of the membrane enzyme diacylglycerol kinase (9) and a similar approach can be used to measure bR thermodynamic stability (10, 11). bR contains a covalently bound retinal chromophore that complicates unfolding analysis because it can slowly hydrolyze off in the SDS unfolded protein (12). We and others originally ...

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Section: Methodsmentioning

confidence: 99%

Measuring membrane protein stability under native conditions

Chang

Bowie

2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Each streptavidin has four biotin binding sites, thereby enabling association of more than one DNA per protein. In order to further control the stoichiometry of biotin on streptavidin, molecularly engineered variants of the protein have been used, which have affinity for only one biotin [74,75] (and hence biotinylated DNA) that permit monolabelling ( per streptavidin molecule) with biotinylated rsfs.royalsocietypublishing.org Interface Focus 6: 20160064 DNA [76]. Molecular cloning has also realized facile production of higher affinity avidin variants such as dimeric rhizavidin [77,78] or electrostatically neutral variants (at physiological pH) such as neutravidin which further expands the tool-kit of this type of conjugation.…”

Section: Affinity For Existing Conjugated Ligandsmentioning

confidence: 99%

Quantum dots–DNA bioconjugates: synthesis to applications

et al. 2016

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Semiconductor nanoparticles particularly quantum dots (QDs) are interesting alternatives to organic fluorophores for a range of applications such as biosensing, imaging and therapeutics. Addition of a programmable scaffold such as DNA to QDs further expands the scope and applicability of these hybrid nanomaterials in biology. In this review, the most important stages of preparation of QD-DNA conjugates for specific applications in biology are discussed. Special emphasis is laid on (i) the most successful strategies to disperse QDs in aqueous media, (ii) the range of different conjugation with detailed discussion about specific merits and demerits in each case, and (iii) typical applications of these conjugates in the context of biology.

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“…Although large nanoparticle labels have also been used for high-resolution tracking of a wide range of membrane proteins (such as glycine receptors (107), GPI anchor proteins (17), a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate receptors (108), integrins (109), and cystic fibrosis transmembrane conductance regulator channel proteins (110,111)), smaller particle labels (diameter < 10 nm) are recently developed and tested for SPT in a confined space (such as synaptic receptors in synaptic cleft (27)), providing less steric hindrance and reduced cross-linking. To completely eliminate cross-linking, we are currently developing monovalent EGFR labeling techniques based on site-specific biotinylation of membrane receptors (113), monovalent streptavidin (114,115), and monovalent nanoparticles (116).…”

Section: Trade-offs In Temporal Resolution and Probe Sizementioning

confidence: 99%

Segmentation of 3D Trajectories Acquired by TSUNAMI Microscope: An Application to EGFR Trafficking

Liu

Perillo

Liu

et al. 2016

Biophysical Journal

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Whereas important discoveries made by single-particle tracking have changed our view of the plasma membrane organization and motor protein dynamics in the past three decades, experimental studies of intracellular processes using singleparticle tracking are rather scarce because of the lack of three-dimensional (3D) tracking capacity. In this study we use a newly developed 3D single-particle tracking method termed TSUNAMI (Tracking of Single particles Using Nonlinear And Multiplexed Illumination) to investigate epidermal growth factor receptor (EGFR) trafficking dynamics in live cells at 16/43 nm (xy/z) spatial resolution, with track duration ranging from 2 to 10 min and vertical tracking depth up to tens of microns. To analyze the long 3D trajectories generated by the TSUNAMI microscope, we developed a trajectory analysis algorithm, which reaches 81% segment classification accuracy in control experiments (termed simulated movement experiments). When analyzing 95 EGF-stimulated EGFR trajectories acquired in live skin cancer cells, we find that these trajectories can be separated into three groups-immobilization (24.2%), membrane diffusion only (51.6%), and transport from membrane to cytoplasm (24.2%). When EGFRs are membrane-bound, they show an interchange of Brownian diffusion and confined diffusion. When EGFRs are internalized, transitions from confined diffusion to directed diffusion and from directed diffusion back to confined diffusion are clearly seen. This observation agrees well with the model of clathrin-mediated endocytosis.

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A monovalent streptavidin with a single femtomolar biotin binding site

Cited by 351 publications

References 30 publications

Measuring membrane protein stability under native conditions

Measuring membrane protein stability under native conditions

Quantum dots–DNA bioconjugates: synthesis to applications

Segmentation of 3D Trajectories Acquired by TSUNAMI Microscope: An Application to EGFR Trafficking

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