An Enhanced System for Unnatural Amino Acid Mutagenesis in E. coli

Young, Travis S.; Ahmad, Insha; Yin, Jun; Schultz, Peter G.

doi:10.1016/j.jmb.2009.10.030

Cited by 576 publications

(755 citation statements)

References 53 publications

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“…This can be explained through azide degradation on storage. Azide-to-amine reduction is reported to be triggered in the presence of Tris[2-carboxyethyl] phosphine (TCEP) as reductant 49 , which was added to maintain the thiol of the cysteine in its reduced form. After functionalization with the dye (for example, Hcp1_cys3_D11pAzF-COMBO-rhod) the protein-adapter could successfully be attached to AuNP of various sizes.…”

Section: Resultsmentioning

confidence: 99%

Molecular protein adaptor with genetically encoded interaction sites guiding the hierarchical assembly of plasmonically active nanoparticle architectures

et al. 2015

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The control over the defined assembly of nano-objects with nm-precision is important to create systems and materials with enhanced properties, for example, metamaterials. In nature, the precise assembly of inorganic nano-objects with unique features, for example, magnetosomes, is accomplished by efficient and reliable recognition schemes involving protein effectors. Here we present a molecular approach using protein-based 'adaptors/ connectors' with genetically encoded interaction sites to guide the assembly and functionality of different plasmonically active gold nanoparticle architectures (AuNP). The interaction of the defined geometricaly shaped protein adaptors with the AuNP induces the self-assembly of nanoarchitectures ranging from AuNP encapsulation to one-dimensional chain-like structures, complex networks and stars. Synthetic biology and bionanotechnology are applied to co-translationally encode unnatural amino acids as additional site-specific modification sites to generate functionalized biohybrid nanoarchitectures. This protein adaptor-based nanoobject assembly approach might be expanded to other inorganic nano-objects creating biohybrid materials with unique electronic, photonic, plasmonic and magnetic properties.

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Section: Resultsmentioning

confidence: 99%

Molecular protein adaptor with genetically encoded interaction sites guiding the hierarchical assembly of plasmonically active nanoparticle architectures

et al. 2015

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“…Arg mutant constructs were cloned via quick-change mutagenesis from the LAP1 LD and LULL1 LD constructs previously described (25). The 293T cells were grown and transfected as previously described (25).…”

Section: Methodsmentioning

confidence: 99%

“…For crosslinking experiments with co-expressed proteins, TorAΔ51 and LAP1 LD or LULL1 LD were expressed in BL21 Star (DE3) cells (Life Technologies) and co-purified by Ni-NTA purification in 2 mM ATP. Crosslinker pBPA was incorporated into TorA-Δ51, LAP1 LD , or LULL1 LD at the indicated amino acid positions, as previously described (25). Cross-linking was performed by incubating reactions at 30°C for 5 min, cooling to 20°C, and UV-irradiating for 15 min.…”

Section: Methodsmentioning

confidence: 99%

“…Analytical gel filtration was performed as described previously (25), except that 7 μM LAP1 LD , LULL1 LD , or their mutants was incubated with an equal concentration of TorA E171Q and 2 mM ATP at 30°C for 5 min before gel filtrations. Fractions were collected and analyzed by SDS/PAGE and immunoblotting.…”

Section: Methodsmentioning

confidence: 99%

“…phenylalanine (BPA) (25) at one of the three adjacent positions on TorA's C-terminal alpha-helix that are expected to contribute to the interface ( Fig. 3 A and B).…”

Section: Significancementioning

confidence: 99%

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The mechanism of Torsin ATPase activation

Brown

Zhao

Chase

et al. 2014

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

146

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Torsins are membrane-associated ATPases whose activity is dependent on two activating cofactors, lamina-associated polypeptide 1 (LAP1) and luminal domain-like LAP1 (LULL1). The mechanism by which these cofactors regulate Torsin activity has so far remained elusive. In this study, we identify a conserved domain in these activators that is predicted to adopt a fold resembling an AAA+ (ATPase associated with a variety of cellular activities) domain. Within these domains, a strictly conserved Arg residue present in both activating cofactors, but notably missing in Torsins, aligns with a key catalytic Arg found in AAA+ proteins. We demonstrate that cofactors and Torsins associate to form heterooligomeric assemblies with a defined Torsin-activator interface. In this arrangement, the highly conserved Arg residue present in either cofactor comes into close proximity with the nucleotide bound in the neighboring Torsin subunit. Because this invariant Arg is strictly required to stimulate Torsin ATPase activity but is dispensable for Torsin binding, we propose that LAP1 and LULL1 regulate Torsin ATPase activity through an active site complementation mechanism.DYT1 dystonia | nuclear envelope | ATPase | Torsin T orsin ATPases belong to the AAA+ (ATPase associated with a variety of cellular activities) superfamily of ATPases (1) but are unusual in that they lack conserved catalytic residues that are typically found in related ATPases (2, 3). Accordingly, Torsins do not display ATPase activity unless they are engaged by their regulatory cofactors lamina-associated polypeptide 1 (LAP1) or luminal domain-like LAP1 (LULL1) (4), which are type II transmembrane proteins residing in the nuclear envelope and endoplasmic reticulum (ER) (5, 6). This property stands in sharp contrast to the behavior of closely related Clp/Hsp100 ATPases, which display considerable basal ATPase activities that are moderately stimulated by their substrates (7,8). Although a number of Clp/Hsp100 proteins use distinct cofactors that confer substrate specificity to the typically hexameric ATPase ring, they operate via similar principles (9). Substrates ultimately engage the pore at the center of the oligomeric assembly. This narrow annulus is defined by pore loops that emanate from each subunit and harbor a conserved aromatic residue that defines the center of the pore (10). The energy of ATP hydrolysis is invested in threading the substrate through this axial channel, and the substrate is unfolded in the process (11).Much less is known about the mode of action of Torsin ATPases, although it is clear that regulation of Torsin activity is of vital importance in an organismal context. A mutation in TorsinA (TorA) causing the movement disorder primary dystonia (12) renders TorA unresponsive to its binding partners LAP1 and LULL1 (4), and a homozygous "knock-in" of this disease allele in mice causes a lethal phenotype, as does a TorA KO (13). A conditional deletion of TorA from the CNS in mice accurately replicates the symptoms of primary dystonia (14). In add...

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Analysis of Protein Ligand‐Receptor Binding by Photoaffinity Cross‐Linking

2015

CP Protein Science

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Photoaffinity cross-linking is a rapidly developing technology for studying biomolecular interactions, including protein ligand-receptor binding. This technology provides detailed binding information including receptor contact sites, active conformation of receptor-ligand complexes, global binding surfaces, and binding modes. Advancements in genetic technology have enabled non-natural photoactive amino acid derivatives to be incorporated into designer or target proteins, providing a host of new opportunities for manufacturing protein photo-probes while bypassing the traditional peptide or small protein limits of classical chemical synthesis. This unit provides several protocols for performing basic photoaffinity cross-linking and related analyses for applications in ligand-receptor binding and protein-protein interactions.

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An Enhanced System for Unnatural Amino Acid Mutagenesis in E. coli

Cited by 576 publications

References 53 publications

Molecular protein adaptor with genetically encoded interaction sites guiding the hierarchical assembly of plasmonically active nanoparticle architectures

Molecular protein adaptor with genetically encoded interaction sites guiding the hierarchical assembly of plasmonically active nanoparticle architectures

The mechanism of Torsin ATPase activation

Analysis of Protein Ligand‐Receptor Binding by Photoaffinity Cross‐Linking

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