Artificial Ligands of Streptavidin (ALiS): Discovery, Characterization, and Application for Reversible Control of Intracellular Protein Transport

Terai, Takuya; Kohno, Moe; Boncompain, Gaëlle; Sugiyama, Shigeru; Saito, Noriko; Fujikake, Ryo; Ueno, Takahiro; Komatsu, Toshimitsu; Hanaoka, Kenjiro; Okabe, Takayoshi; Urano, Yasuteru; Perez, Franck; Nagano, Tetsuo

doi:10.1021/jacs.5b05672

Cited by 24 publications

(38 citation statements)

References 29 publications

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“…Moreover, these stabilized open L3,4 loops may also expose the biotin binding site, resulting in fast release of biotin. Consistently, the open L3,4 was previously proposed to explain the fast dissociation of ALiS from SA15. All these effects together may account for the substantial reduction of biotin binding to N23A/S27D/S45A mutants12.…”

Section: Discussionsupporting

confidence: 77%

The Crystal Structure of Monovalent Streptavidin

Zhang

Biswas

Deng

et al. 2016

Sci Rep

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The strong interaction between streptavidin (SA) and biotin is widely utilized in biotechnological applications. A SA variant, monovalent SA, was developed with a single and high affinity biotin-binding site within the intact tetramer. However, its structural characterization remains undetermined. Here, we seek to determine the crystal structure of monovalent SA at 1.7-Å resolution. We show that, in contrast to its ‘close-state’ in the only wild-type subunit, the L3,4 loops of three Dead SA subunits are free from crystal packing and remain in an ‘open state’, stabilized by a consistent H-bonding network involving S52. This H-bonding network also applies to the previously reported open state of the wild-type apo-SA. These results suggest that specific substitutions (N23A/S27D/S45A) at biotin-binding sites stabilize the open state of SA L3,4 loop, thereby further reducing biotin-binding affinity. The general features of the ‘open state’ SA among different SA variants may facilitate its rational design. The structural information of monovalent SA will be valuable for its applications across a wide range of biotechnological areas.

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Section: Discussionsupporting

confidence: 77%

The Crystal Structure of Monovalent Streptavidin

Zhang

Biswas

Deng

et al. 2016

Sci Rep

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“…28,30 Moreover, in a buffered aqueous solution, excitation of these tryptophan residues leads to an average emission λ max of 336 nm that can initiate FRET with biotinylated fluorophores. 20,31,32 Correspondingly, docking 33 of this X-ray structure (Figure 3) to PB-biotin derivative 12 (Figure 4, panel A) supported the notion that binding of SA would favorably position the fluorophore in close proximity to endogenous tryptophan residues that make noncovalent contacts to biotin and tryptophan residues in other subunits of the SA tetramer.…”

Section: Resultssupporting

confidence: 60%

Quantification of Small Molecule–Protein Interactions using FRET between Tryptophan and the Pacific Blue Fluorophore

Lee

Peterson

2016

ACS Omega

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We report a new method to quantify the affinity of small molecules for proteins. This method is based on Förster resonance energy transfer (FRET) between endogenous tryptophan (Trp) residues and the coumarin-derived fluorophore Pacific Blue (PB). Tryptophan residues are frequently found in proteins near ligand-binding sites, making this approach potentially applicable to a wide range of systems. To improve access to PB, we developed a scalable multigram synthesis of this fluorophore, starting with inexpensive 2,3,4,5-tetrafluorobenzoic acid. This route was used to synthesize fluorescent derivatives of biotin, as well as lower affinity thiobiotin, iminobiotin, and imidazolidinethione analogues that bind the protein streptavidin. Compared with previously published FRET acceptors for tryptophan, PB proved to be superior in both sensitivity and efficiency. These unique properties of PB enabled direct quantification of dissociation constants (Kd) as well as competitive inhibition constants (Ki) in the micromolar to nanomolar range. In comparison to analogous binding studies using fluorescence polarization, fluorescence quenching, or fluorescence enhancement, affinities determined using Trp-FRET were more precise and accurate as validated using independent isothermal titration calorimetry studies. FRET between tryptophan and PB represents a new tool for the characterization of protein–ligand complexes.

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“…Ω for serum albumin (PDB: 4F5S), 43 streptavidin (PDB: 4Y5D), 44 avidin (PDB: 1AVD), 45 and alcohol dehydrogenase (PDB: 5ENV) 46 were calculated using the projection approximation 47 and exact hard-sphere scattering 48 methods as implemented in EHSS2/k. 49 Prior to the Ω calculations, all noncovalent additions to the protein(s) in the deposited structures, including water molecules and metal ions, were deleted and Chimera 50 was used to complete side chains and add hydrogen atoms.…”

Section: Methodsmentioning

confidence: 99%

Interpreting the Collision Cross Sections of Native-like Protein Ions: Insights from Cation-to-Anion Proton-Transfer Reactions

Laszlo

Bush

2017

Anal. Chem.

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The effects of charge state on structures of native-like cations of serum albumin, streptavidin, avidin, and alcohol dehydrogenase were probed using cation-to-anion proton-transfer reactions (CAPTR), ion mobility, mass spectrometry, and complementary energy-dependent experiments. The CAPTR products all have collision cross section (Ω) values that are within 5.5% of the original precursor cations. The first CAPTR event for each precursor yields products that have smaller Ω values and frequently exhibit the greatest magnitude of change in Ω resulting from a single CAPTR event. To investigate how the structures of the precursors affect the structures of the products, ions were activated as a function of energy prior to CAPTR. In each case, the Ω of the activated precursors increase with increasing energy, but the Ω of the CAPTR products are smaller than the activated precursors. To investigate the stabilities of the CAPTR products, the products were activated immediately prior to ion mobility. These results show that additional structures with smaller or larger Ω can be populated and that the structures and stabilities of these ions depend most strongly on the identity of the protein and the charge state of the product, rather than the charge state of the precursor or the number of CAPTR events. Together, these results indicate that the excess charges initially present on native-like ions have a modest, but sometimes statistically significant, effect on their Ω values. Therefore, potential contributions from charge state should be considered when using experimental Ω values to elucidate structures in solution.

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Artificial Ligands of Streptavidin (ALiS): Discovery, Characterization, and Application for Reversible Control of Intracellular Protein Transport

Cited by 24 publications

References 29 publications

The Crystal Structure of Monovalent Streptavidin

The Crystal Structure of Monovalent Streptavidin

Quantification of Small Molecule–Protein Interactions using FRET between Tryptophan and the Pacific Blue Fluorophore

Interpreting the Collision Cross Sections of Native-like Protein Ions: Insights from Cation-to-Anion Proton-Transfer Reactions

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